An extracellular bacterial pathogen modulates host metabolism to regulate its own sensing and proliferation.

Successful infection depends on the ability of the pathogen to gain nutrients from the host. The extracellular pathogenic bacterium group A Streptococcus (GAS) causes a vast array of human diseases. By using the quorum-sensing sil system as a reporter, we found that, during adherence to host cells, GAS delivers streptolysin toxins, creating endoplasmic reticulum stress. This, in turn, increases asparagine (ASN) synthetase expression and the production of ASN. The released ASN is sensed by the bacteria, altering the expression of ∼17% of GAS genes of which about one-third are dependent on the two-component system TrxSR. The expression of the streptolysin toxins is strongly upregulated, whereas genes linked to proliferation are downregulated in ASN absence. Asparaginase, a widely used chemotherapeutic agent, arrests GAS growth in human blood and blocks GAS proliferation in a mouse model of human bacteremia. These results delineate a pathogenic pathway and propose a therapeutic strategy against GAS infections.

Genetic and Structural Analyses of RRNPP Intercellular Peptide Signaling of Gram-Positive Bacteria.

Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure-function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.

Bacterial second messenger cyclic di-AMP in streptococci.

Cyclic dimeric adenosine monophosphate (c-di-AMP) has been well studied in bacteria, including those of the genus Streptococcus, since the first recognition of this dinucleotide in 2008. Streptococci possess a sole diadenylate cyclase, CdaA, and distinct c-di-AMP phosphodiesterases. Interestingly, cdaA is required for viability of some streptococcal species but not all when streptococci are grown in standard laboratory media. Bacteria of this genus also have distinct c-di-AMP effector proteins, diverse c-di-AMP-signaling pathways, and subsequent biological outcomes. In streptococci, c-di-AMP may influence bacterial growth, morphology, biofilm formation, competence program, drug resistance, and bacterial pathogenesis. c-di-AMP secreted by streptococci has also been shown to interact with the mammalian host and induces immune responses including type I interferon production. In this review, we summarize the reported c-di-AMP networks in seven species of the genus Streptococcus, which cause diverse clinical manifestations, and propose future perspectives to investigate the signaling molecule in these streptococcal pathogens.

Clinical isolates of Streptococcus mitis/oralis-related species with reduced carbapenem susceptibility, harboring amino acid substitutions in penicillin-binding proteins in Japan.

Streptococcus mitis/oralis group isolates with reduced carbapenem susceptibility have been reported, but its isolation rate in Japan is unknown. We collected 356 clinical α-hemolytic streptococcal isolates and identified 142 of them as S. mitis/oralis using partial sodA sequencing. The rate of meropenem non-susceptibility was 17.6% (25/142). All 25 carbapenem-non-susceptible isolates harbored amino acid substitutions in/near the conserved motifs in PBP1A, PBP2B, and PBP2X. Carbapenem non-susceptibility is common among S. mitis/oralis group isolates in Japan.

Combined pH ratiometry and fluorescence lectin-binding analysis (pH-FLBA) for microscopy-based analyses of biofilm pH and matrix carbohydrates.

Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH (P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates (P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.IMPORTANCEBiofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid-base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.

Bicyclostreptins are radical SAM enzyme-modified peptides with unique cyclization motifs.

Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins-peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic ß-ether and a heterocyclic sp3-sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci.

The ComRS-SigX Pathway Regulates Natural Transformation in Streptococcus ferus.

The ability to take up and incorporate foreign DNA via natural transformation is a well-known characteristic of some species of Streptococcus, and is a mechanism that rapidly allows for the acquisition of antibacterial resistance. Here, we describe that the understudied species Streptococcus ferus is also capable of natural transformation and uses a system analogous to that identified in Streptococcus mutans. S. mutans natural transformation is under the control of the alternative sigma factor sigX (also known as comX), whose expression is induced by two types of peptide signals: CSP (competence stimulating peptide, encoded by comC) and XIP (sigX-inducing peptide, encoded by comS). These systems induce competence via either the two-component signal-transduction system ComDE or the RRNPP transcriptional regulator ComR, respectively. Protein and nucleotide homology searches identified putative orthologs of comRS and sigX in S. ferus, but not homologs of S. mutans blpRH (also known as comDE). We demonstrate that natural transformation in S. ferus is induced by a small, double-tryptophan containing sigX-inducing peptide (XIP), akin to that of S. mutans, and requires the presence of the comR and sigX orthologs for efficient transformation. Additionally, we find that natural transformation is induced in S. ferus by both the native XIP and the XIP variant of S. mutans, implying that cross talk between the two species is possible. This process has been harnessed to construct gene deletions in S. ferus and provides a method to genetically manipulate this understudied species. IMPORTANCE Natural transformation is the process by which bacteria take up DNA and allows for acquisition of new genetic traits, including those involved in antibiotic resistance. This study demonstrates that the understudied species Streptococcus ferus is capable of natural transformation using a peptide-pheromone system like that previously identified in Streptococcus mutans and provides a framework for future studies concerning this organism.

Analysing the dynamics of the bacterial community in pozol, a Mexican fermented corn dough.

Pozol is a traditional prehispanic Mexican beverage made from fermented nixtamal dough; it is still part of everyday life in many communities due to its nutritional properties. It is the product of spontaneous fermentation and has a complex microbiota composed primarily of lactic acid bacteria (LAB). Although this is a beverage that has been used for centuries, the microbial processes that participate in this fermented beverage are not well understood. We fermented corn dough to produce pozol and sampled it at four key times to follow the community and metabolic changes (0, 9 24 and 48 h) by shotgun metagenomic sequencing to determine structural changes in the bacterial community, as well as metabolic genes used for substrate fermentation, nutritional properties and product safety. We found a core of 25 abundant genera throughout the 4 key fermentation times, with the genus Streptococcus being the most prevalent throughout fermentation. We also performed an analysis focused on metagenomic assembled genomes (MAGs) to identify species from the most abundant genera. Genes involving starch, plant cell wall (PCW), fructan and sucrose degradation were found throughout fermentation and in MAGs, indicating the metabolic potential of the pozol microbiota to degrade these carbohydrates. Complete metabolic modules responsible for amino acid and vitamin biosynthesis increased considerably during fermentation, and were also found to be abundant in MAG, highlighting the bacterial contribution to the well-known nutritional properties attributed to pozol. Further, clusters of genes containing CAZymes (CGCs) and essential amino acids and vitamins were found in the reconstructed MAGs for abundant species in pozol. The results of this study contribute to our understanding of the metabolic role of micro-organisms in the transformation of corn to produce this traditional beverage and their contribution to the nutritional impact that pozol has had for centuries in the traditional cuisine of southeast Mexico.

A second riboswitch class for the enzyme cofactor NAD.

A bacterial noncoding RNA motif almost exclusively associated with pnuC genes was uncovered using comparative sequence analysis. Some PnuC proteins are known to transport nicotinamide riboside (NR), which is a component of the ubiquitous and abundant enzyme cofactor nicotinamide adenine dinucleotide (NAD+). Thus, we speculated that the newly found "pnuC motif" RNAs might function as aptamers for a novel class of NAD+-sensing riboswitches. RNA constructs that encompass the conserved nucleotides and secondary structure features that define the motif indeed selectively bind NAD+, nicotinamide mononucleotide (NMN), and NR. Mutations that disrupt strictly conserved nucleotides of the aptamer also disrupt ligand binding. These bioinformatic and biochemical findings indicate that pnuC motif RNAs are likely members of a second riboswitch class that regulates gene expression in response to NAD+ binding.

A novel sialic acid-binding adhesin present in multiple species contributes to the pathogenesis of Infective endocarditis.

Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP- isolates also bind the cryptic receptor ß-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.

Esculetin improves murine mastitis induced by streptococcus isolated from bovine mammary glands by inhibiting NF-κB and MAPK signaling pathways.

Cow mastitis, caused by Streptococcus infection of the mammary glands, is a common clinical disease that can lead to decreased milk quality and threaten animal welfare and performance. Esculetin (ESC) is a coumarin with anti-inflammatory and anti-asthmatic effects. However, whether ESC has therapeutic effects on mastitis remains unexplored. This study was conducted to investigate the protective effect of ESC against murine mastitis caused by Streptococcus isolated from bovine mammary glands and elucidate the underlying mechanisms. Streptococcus uberis was used to construct a mouse model of mastitis. The results showed that the mice exhibited edema and thickening of the acinar wall with inflammatory infiltration after S. uberis treatment. Intraperitoneal injection of ESC significantly reduced inflammatory cell infiltration, restored normal physiological function, and inhibited the production of the inflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot analysis revealed that ESC reduced P38 phosphorylation, further inhibited the influence of mammary Streptococcus on cytoplasmic translocation of nuclear factor-κB (P65), and inhibited the transcriptional activation of P65, thus inhibiting the generation of inflammatory cells. Collectively, ESC may inhibit mitogen-activated protein kinase and nuclear factor-κB, thereby highlighting its potential for the treatment and prevention of mastitis.

Molecular dissection of pheromone selectivity in the competence signaling system ComRS of streptococci.

Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence traits. In most streptococci, competence is regulated by ComRS signaling, a system based on the mature ComS pheromone (XIP), which is internalized to activate the (R)RNPP-type ComR sensor by triggering dimerization and DNA binding. Cross-talk analyses demonstrated major differences of selectivity between ComRS systems and raised questions concerning the mechanism of pheromone-sensor recognition and coevolution. Here, we decipher the molecular determinants of selectivity of the closely related ComRS systems from Streptococcus thermophilus and Streptococcus vestibularis Despite high similarity, we show that the divergence in ComR-XIP interaction does not allow reciprocal activation. We perform the structural analysis of the ComRS system from S. vestibularis. Comparison with its ortholog from S. thermophilus reveals an activation mechanism based on a toggle switch involving the recruitment of a key loop by the XIP C terminus. Together with a broad mutational analysis, we identify essential residues directly involved in peptide binding. Notably, we generate a ComR mutant that displays a fully reversed selectivity toward the heterologous pheromone with only five point mutations, as well as other ComR variants featuring XIP bispecificity and/or neofunctionalization for hybrid XIP peptides. We also reveal that a single XIP mutation relaxes the strictness of ComR activation, suggesting fast adaptability of molecular communication phenotypes. Overall, this study is paving the way toward the rational design or directed evolution of artificial ComRS systems for a range of biotechnological and biomedical applications.

Structure-function studies of Rgg binding to pheromones and target promoters reveal a model of transcription factor interplay.

Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryoelectron microscopy (cryo-EM) structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures with that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both Streptococcus dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.

Increased IL-12p70 and IL-8 Produced by Monocytes in Response to Streptococcus spp. and Actinomyces spp. Causals of Endodontic Primary Infections.

We sought to evaluate the effect of endodontic-causative microorganisms of primary infections on mononuclear cells such as CD14+, CD4+, CD8+, CD19+ and Tregs Foxp3+. Facultative anaerobic microorganisms were isolated from radicular conducts and peripheral blood samples, which were taken from patients with primary infections. Cellular cultures were performed with peripheral blood mononuclear cells (PBMC) with and without Actinomyces spp. and Streptococcus spp. during 48, 72, and 96 h of contact in culture (concentration 5 × 105 cells/well) in a round plate bound with 48 wells. Later, PBMC was collected for analysis by flow cytometry, with the monoclonal antibodies αCD14, αCD4, αCD8, αCD19 and αFoxp3, and acquired using an FACSCanto II cytometer. The supernatant of cellular cultures was analyzed for the quantification of inflammatory cytokines. Data analysis was performed in FlowJo v10.8.2 and FCAPArray software, and statistical analysis was performed using GraphPad v5.0. software. We observed an increase in the percentage of CD14+ cells in patients at different hours of cellular culture in the presence of both Actinomyces spp. and Streptococcus spp. microorganisms, compared to healthy controls. This study demonstrates the role played by the innate immune system in the pathogeny of endodontic primary infections, explaining the effects that generate the more common microorganisms in this oral pathology.

Oral Commensal Streptococci: Gatekeepers of the Oral Cavity.

Oral commensal streptococci are primary colonizers of the oral cavity. These streptococci produce many adhesins, metabolites, and antimicrobials that modulate microbial succession and diversity within the oral cavity. Often, oral commensal streptococci antagonize cariogenic and periodontal pathogens such as Streptococcus mutans and Porphyromonas gingivalis, respectively. Mechanisms of antagonism are varied and range from the generation of hydrogen peroxide, competitive metabolite scavenging, the generation of reactive nitrogen intermediates, and bacteriocin production. Furthermore, several oral commensal streptococci have been shown to alter the host immune response at steady state and in response to oral pathogens. Collectively, these features highlight the remarkable ability of oral commensal streptococci to regulate the structure and function of the oral microbiome. In this review, we discuss mechanisms used by oral commensal streptococci to interact with diverse oral pathogens, both physically and through the production of antimicrobials. Finally, we conclude by exploring the critical roles of oral commensal streptococci in modulating the host immune response and maintaining health and homeostasis.

Oxidative Reactions Catalyzed by Hydrogen Peroxide Produced by Streptococcus pneumoniae and Other Streptococci Cause the Release and Degradation of Heme from Hemoglobin.

Streptococcus pneumoniae (Spn) strains cause pneumonia that kills millions every year worldwide. Spn produces Ply, a hemolysin that lyses erythrocytes releasing hemoglobin, and also produces the pro-oxidant hydrogen peroxide (Spn-H2O2) during growth. The hallmark of the pathophysiology of hemolytic diseases is the oxidation of hemoglobin, but oxidative reactions catalyzed by Spn-H2O2 have been poorly studied. We characterized the oxidation of hemoglobin by Spn-H2O2. We prepared a series of single-mutant (ΔspxB or ΔlctO), double-mutant (ΔspxB ΔlctO), and complemented strains in TIGR4, D39, and EF3030. We then utilized an in vitro model with oxyhemoglobin to demonstrate that oxyhemoglobin was oxidized rapidly, within 30 min of incubation, by Spn-H2O2 to methemoglobin and that the main source of Spn-H2O2 was pyruvate oxidase (SpxB). Moreover, extended incubation caused the release and the degradation of heme. We then assessed oxidation of hemoglobin and heme degradation by other bacterial inhabitants of the respiratory tract. All hydrogen peroxide-producing streptococci tested caused the oxidation of hemoglobin and heme degradation, whereas bacterial species that produce <1 µM H2O2 neither oxidized hemoglobin nor degraded heme. An ex vivo bacteremia model confirmed that oxidation of hemoglobin and heme degradation occurred concurrently with hemoglobin that was released from erythrocytes by Ply. Finally, gene expression studies demonstrated that heme, but not red blood cells or hemoglobin, induced upregulated transcription of the spxB gene. Oxidation of hemoglobin may be important for pathogenesis and for the symbiosis of hydrogen peroxide-producing bacteria with other species by providing nutrients such as iron.

Select Streptococci Can Degrade Candida Mannan To Facilitate Growth.

Multiple studies have found that streptococci have a synergistic relationship with Candida species, but the details of these interactions are still being discovered. Candida species are covered by mannan, a polymer of mannose, which could serve as a carbon source for certain microbes. We hypothesized that streptococci that possess mannan-degrading glycosyl hydrolases would be able to enzymatically cleave mannose residues, which could serve as a primary carbohydrate source to support growth. We analyzed 90 streptococcus genomes to predict the capability of streptococci to transport and utilize mannose and to degrade diverse mannose linkages found on mannan. The genome analysis revealed mannose transporters and downstream pathways in most streptococci, but only <50% of streptococci harbored the glycosyl hydrolases required for mannan degradation. To confirm the ability of streptococci to use mannose or mannan, we grew 6 representative streptococci in a chemically defined medium lacking glucose supplemented with mannose, yeast extract, or purified mannan isolated from Candida and Saccharomyces strains. Although all tested Streptococcus strains could use mannose, Streptococcus salivarius and Streptococcus agalactiae, which did not possess mannan-degrading glycosyl hydrolases, could not use yeast extract or mannan to enhance their growth. In contrast, we found that Streptococcus mitis, Streptococcus parasanguinis, Streptococcus sanguinis, and Streptococcus pyogenes possessed the necessary glycosyl hydrolases to use yeast extract and isolated mannan, which promoted robust growth. Our data indicate that several streptococci are capable of degrading fungal mannans and harvesting mannose for energy. IMPORTANCE This work highlights a previously undescribed aspect of streptococcal Candida interactions. Our work identifies that certain streptococci possess the enzymes required to degrade mannan, and through this mechanism, they can release mannose residues from the cell wall of fungal species and use them as a nutrient source. We speculate that streptococci that can degrade fungal mannan may have a competitive advantage for colonization. This finding has broad implications for human health, as streptococci and Candida are found at multiple body sites.

Oxidative dehalogenation and mineralization of polychlorinated biphenyls by a resuscitated strain Streptococcus sp. SPC0.

Most functional microorganisms cannot be cultivated due to entering a viable but non-culturable (VBNC) state, which limits the characterization and application of polychlorinated biphenyl (PCB)-degrading strains. Resuscitating VBNC bacteria could provide huge candidates for obtaining high-efficient PCB degraders. However, limited studies have focused on the ability of resuscitated strains for PCBs degradation. In the present study, whole-genome analysis of a resuscitated strain SPC0, and its performances in degradation of three prevalent PCB congeners (PCBs 18, 52 and 77) were investigated. The results indicate that the strain SPC0 belonged to the genus Streptococcus, possessed the degradation potential for aromatic xenobiotics. The SPC0 could effectively degrade PCBs 18 and 52, but exhibited lower degradation efficiency of PCB 77. Degradation of PCBs 18 and 52 could be fitted well by zero-order model, whereas the fittest model for PCB 77 degradation was pseudo second-order kinetics. The bph genes expression, chloride ions release and degradation metabolites identification, suggest that SPC0 possessed the capability of oxidative dehalogenation and mineralization of PCBs. Interestingly, SPC0 can degrade PCBs via the bph-encoded biphenyl pathway, and further mineralize metabolite dichlorobenzoate via protocatechuate pathway. This study is the first to show that a strain belonging to genus Streptococcus possessed PCB-degrading capability, which uncovered the powerful potential of resuscitated strains for bioremediation of PCB-contaminated sites.

Comparative Proteomic Analysis of Transcriptional and Regulatory Proteins Abundances in S. lividans and S. coelicolor Suggests a Link between Various Stresses and Antibiotic Production.

Streptomyces coelicolor and Streptomyces lividans constitute model strains to study the regulation of antibiotics biosynthesis in Streptomyces species since these closely related strains possess the same pathways directing the biosynthesis of various antibiotics but only S. coelicolor produces them. To get a better understanding of the origin of the contrasted abilities of these strains to produce bioactive specialized metabolites, these strains were grown in conditions of phosphate limitation or proficiency and a comparative analysis of their transcriptional/regulatory proteins was carried out. The abundance of the vast majority of the 355 proteins detected greatly differed between these two strains and responded differently to phosphate availability. This study confirmed, consistently with previous studies, that S. coelicolor suffers from nitrogen stress. This stress likely triggers the degradation of the nitrogen-rich peptidoglycan cell wall in order to recycle nitrogen present in its constituents, resulting in cell wall stress. When an altered cell wall is unable to fulfill its osmo-protective function, the bacteria also suffer from osmotic stress. This study thus revealed that these three stresses are intimately linked in S. coelicolor. The aggravation of these stresses leading to an increase of antibiotic biosynthesis, the connection between these stresses, and antibiotic production are discussed.

A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2'-OH of GTP.

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3'-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 2'-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogs substituted with different chemical moieties at the 2' position suggest a clear role for 2'-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2'-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.